The measurement of the ratio of the forward-propagating (“F”) to backward propagating (“B”) SHG signal (the “F/B ratio”) has reportedly been used to study collagen fiber ordering in various tissue samples. The F/B ratio revealed the length scale of ordering in the fibers, and in the case of osteogenesis imperfecta and ovarian cancer, was able to be used to discriminate pathological tissue from healthy tissue. This suggests that this technique might serve as a diagnostic tool for these diseases and possibly others in the future.
Previously reported SHG F/B ratio measurements were made in vitro, because a second objective lens was needed to collect forward propagating SHG signal and thus the tissue sample had to be dissected from the subject and sectioned to allow signal to reach the second detection lens. For clinical applications, as well as basic science applications in the in vivo setting, these requirements are problematic.
In Han et al., Measurement of the ratio of forward-propagating to back propagating second harmonic signal using a single objective, Optics Express, 2010. 18(10): p. 10538-10550, the instant [means ‘current’ in patentees] inventors reported on a SHG confocal imaging method that could produce F/B ratio measurements without a second lens (to collect the “F” SHG light) on the opposite side of the sample. The method involved taking SHG images over the same region of interest repeatedly through a series of confocal pinholes of different sizes. It allowed the successful measurement of rat tail collagen [not necessary for ‘this’ application] SHG F/B ratio in vivo, i.e., while the collagen fibers were still embedded in the tail. To the inventors' knowledge, this variable pinhole method was the first available imaging technique that allowed one to measure the SHG F/B ratio on intact tissue samples without sectioning, using just an epi-detection objective lens.
In the aforementioned variable pinhole method, five SHG images were taken over the same region of interest, which is a time consuming process. As a consequence, the sample must be kept stationary during the whole imaging process to avoid SHG intensity variation due to the movement of the sample. During in vivo applications, applications on extremely thick and soft tissues such as, but not limited to, tissue freshly removed from a subject during surgery, and in endoscopic use applications, for example, this can be problematic.
In view of the foregoing discussion of the shortcomings and problems associated with measuring the SHG F/B ratio on intact tissue samples without sectioning using just an epi-detection objective lens, such as but not limited to sample motion, the inventors have recognized the benefits and advantages of apparatus and methods that overcome such shortcomings and solve said problems.
In breast conservation surgery (BCS) the surgeon attempts to remove the primary breast tumor, plus a sufficient margin such that no tumor cells remain in the patient, while leaving as much of the breast untouched as possible. The surgeon can often take advantage of optical and tactile cues while performing the surgery, as well as radiographic imaging of the excised tissue, to deduce whether sufficient tissue has been removed to ensure that margins are clear, which is usually defined as no tumor cells within ˜2 mm of the excised tissue surface. This is supported by subsequent pathology investigation, in which the excised tissue is thinly sliced, stained, and coarsely subsampled at high resolution by a pathologist, who determines the location of tumor cells relative to the surgical margin in those slides that are sampled. This assessment by the pathologist is especially important in tumors such as DCIS in which optical, tactile, and radiographic cues are minimal. Unfortunately, the pathologist is not able to look, at high magnification, at every cubic micron of excised tissue within 2 mm of the excised surface, is therefore forced to perform a subsampling, and hence can miss aberrant tissue, contributing to the number of patients whose tumors recur in spite of reportedly good margins. This negatively impacts patient outcomes. Furthermore, full assessment takes significantly more time than the duration of the surgical procedure, and patients must therefore return to the hospital for second and third surgeries when the conclusion is reached that their samples had poor margins. This negatively impacts patient quality of life.
In view thereof, it would be advantageous to develop a method for rapid margin assessment in the operating room, particularly applicable to intact (i.e. unsliced) tissue section typically removed during breast conservation surgery.